Growing xenotransplant material in culture

ABSTRACT

Over time, cultured mammalian cells aggregate into an artificial tissue with 10% homologous serum and nicotinamide. Neonatal porcine islets alone have a SGS index of 4.6. Co-culture with neonatal porcine Sertoli cells formed free-floating islet-like structures, 300-600 microns in diameter (SGS index 21). Basal secretion of insulin remains high for islets in a heavily confluent mixed cell culture. Re-seeded cultures were very responsive to glucose (SGS index 21.8). Co-culture with fibroblasts also provides a high SGS index, permitting use of the recipient&#39;s fibroblast line as support/trophic cells for an xenotransplantable graft, as for treatment of diabetes.

FIELD

This invention generally relates to growing biological material bycell/tissue culture methods in order to make material suitable fortransplanting into a mammal in order to overcome a disease, deficiency,or defect. More particularly the invention relates to preparations ofcells and cell combinations for use in xenotransplantation, andspecifically the invention relates to transplants of endocrine secretorycells such as islet cells for treating diabetes present within therecipient.

BACKGROUND

A patient afflicted with the irreversible disease diabetes has defectiveislets of Langerhans and cannot produce sufficient insulin to respondappropriately to blood glucose increase. Since the discovery of insulinby Banting et al in 1922, exogenous (injected) insulin in repeated doseshas comprised an effective method of control of blood glucose and hasgreatly extended the lives of many diabetics. Implants for use incontrolling endogenous secretion defects such as diabetes are known,where ideally the implant would (a) produce sufficient insulin to avoidany need for use of injections or other means of administration ofinsulin, and also (b) assume a regulatory role, responding to swings inblood glucose concentration by producing a greater amount of insulin ifthe environment of the implant has an increased concentration ofglucose. Kidney and pancreas, or pancreas alone allotransplantation isdone from cadaver sources or sometimes live donors, but faces the usualproblems of rejection involving immune responses. Islet transplantationafter enzymic digestion of a cadaver pancreas is also known (e.g.Shapiro et al).

Xenotransplants of islet cells present a number of advantages, such asavoidance of certain diseases. For an xenotransplant, barriers ofvarious types are used at least to confine the cells to a permanentlyidentifiable, often subcutaneous site as well as to avoid an immunesystem challenge from the patient yet not detracting from the intendedfunction. Specific pathogen free (SPF) pigs are suitable donors forxenografts of many types because of their anatomical and physiologicalsimilarities with humans and the porcine pancreas has been considered asa potential source of islets for xenotransplantation in diabeticpatients. However, the high cost of SPF herd maintenance as well as thenecessity to tightly control potential zoonoses has led to efforts topropagate islets in vitro—a kind of organ culture. Growing of animalcells in vitro for xenotransplantation if it were possible would providemany advantages including cost savings, and better control of theproduct, including better control of infectious pathogens such aspotentially zoonotic viruses.

Previous research papers have shown only limited in vitro growth ofadult and neonatal islets of different species. Neonatal tissue has agood capacity for growth in tissue culture, although a cleardisadvantage of neonatal islet tissue is functional immaturity, whichresults in a period of 6-8 weeks before the grafted cells are able toregulate the hyperglycemia in diabetic recipients (see for exampleKorgsen O, Jansson L, Eizirik DL, Andersson A. “Functional andmorphological differentiation of fetal porcine islet-like cells clustersafter transplantation into nude mice”. Diabetologia 1991; 34:379). It isalso relatively difficult to hold differentiated cells (such as isletcells) in culture over long periods without reversion to a poorlydifferentiated version.

There are many other forms of disease where the possibility of insertingextra material into the body in order to overcome the problem exists.For example, replacement of functions not or no longer available fromother endocrine glands, the liver, or muscles or tendons, cartilage asblocks or other shapes for reconstruction, and lumps of bone.

One problem to be solved is to raise potentially insulin-secreting cellsin a culture from, for example, neonatal porcine pancreatic tissue;including the step of maintaining the cells for an extended period.Another problem to be solved is to provide an effective xenotransplantcapable of making an effective amount of insulin when exposed tohyperglycaemic conditions, despite the usual presence of a physicalbarrier intended to limit access of members of the immune system to theforeign cells.

OBJECT

It is an object of this invention to create an improved cell preparationfor use in xenotransplants, or at least to provide the public with auseful choice.

Definitions

homologous serum: a serum for cell culture, taken from the same speciesof animal as that from which the cells were derived.

artificial tissue: a mixture, made de novo of more than one kind ofliving cell in tissue/cell culture, and after a period of timecharacteristically exhibiting (a) at least some physiological support ofone kind of cell by another kind, and (b) at least some distinctivestructural configuration including the cells of the mixture.

connective tissue secretory cells; a group including cells capable ofsecreting and supporting components of an extracellular matrix, such aschondrocytes, osteocytes, fibrocytes, including fibrocytes from tendonsand fibrocytes from the dermis of the skin, and undifferentiatedfibroblasts. This may include stem cells.

endocrine secretory cells: a group including, but not limited to:anterior pituitary cells, thyroid and parathyroid cells, pancreaticislet cells, adrenal cortical cells, ovarian and testicular cellsincluding interstitial cells, choroid plexus cells, and the like.

Other first-type cells (as used herein) include any functional celltypes including those of epithelial, connective tissue, or neural originespecially hepatocytes.

support/trophic cells: includes Sertoli cells, fibroblasts, some typesof neuroglial cells, secretory cells including choroid plexus epithelialcells, and stem cells. Modified support/trophic cells include cellsmodified by alteration of their genetic composition so that they secretea range of compounds similar to those secreted by, for example, Sertolicells in a tissue culture environment and capable of promoting thegrowth and development of functional characteristics of secretory cellswhen grown in association.

long-term: a period of more than a week; typically extended to 4-6 weeksor more.

STATEMENT OF INVENTION

In a first broad aspect this invention provides a method for carryingout long-term in vitro assembly, propagation and maturation of afunctional artificial tissue comprised of more than one kind of celltaken from at least one species of mammal, wherein a selected first typeof cell capable of being stimulated to secrete a product is co-culturedover an extended period together with at least one selected second typeof cell capable of providing a support/trophic function for the firsttype, in a growth medium supplemented with an effective amount ofhomologous serum from the species of mammal that provided the first typeof cell, so that organised functional, synergistic groups of mixed celltypes are developed over the extended period.

In a subsidiary aspect the invention provides a method as previouslydescribed in this section for making a functional artificial tissuewherein the artificial tissue includes a selected first type of cell anda second type of cell jointly capable of possessing a property, and isgrown as a co-culture in a growth media supplemented with an effectiveamount of homologous serum, so that the artificial tissue may be graftedinto a recipient suffering from a deficiency of the property in order toalleviate the deficiency.

Preferably the first type of cell is selected from the group ofendocrine secretory cells (as herein defined) and the product is anendocrine hormone.

Preferably the second type of cell is selected from a group includingSertoli cells and fibroblasts.

Optionally the first type of cell is not an endocrine cell.

In a related aspect, the effective amount of homologous serum is in arange of from about 5% to about 15% by volume in the growth medium.

In a second related aspect, the growth media also includes nicotinamidewithin a range of from about 5 mM to about 15 mM.

Preferably at least the first type of cell is collected from a neonatalmammal.

Preferably, for making a functional artificial tissue for use inalleviation of diabetes, the first type of cell is endocrine cells fromthe pancreatic islets of Langerhans.

More particularly the artificial tissue is grown as a co-culture in agrowth media supplemented with an effective amount of homologous serum,glucose, and nicotinamide, so that the resulting artificial tissueexhibits a functional secretory response to a local concentration ofglucose, so that the artificial tissue may be grafted into a recipientsuffering from diabetes in order to alleviate the disease.

In a third related aspect, the second type of cell is taken by biopsyfrom an individual in need of a functional graft and after in vitropropagation and maturation together with the first type of cell for aperiod the resulting co-culture is returned to the diabetic recipient inthe form of a functional artificial tissue.

Preferably the second-type cells are fibroblasts, obtained from aspecific individual intended as a subject for a subsequent xenograftingprocedure in which the individual receives subsequent generations of thefibroblasts as an allograft.

Typically, functional cell combinations have been grown as a co-culturefor from about 30 to about 50 days in growth media of this type.

In a second broad aspect, the invention provides a functional artificialtissue comprised of more than one kind of cell taken from at least onespecies of mammal, wherein the functional artificial tissue is comprisedof at least partially organised synergistic accumulations of cellscomprised of a first type of cell, capable of being stimulated tosecrete a product, and at least one second type of cell capable ofproviding a support/trophic function for the first type of cell, thefunctional artificial tissue being formed by co-culture in vitro over anextended period of time.

Preferably the first type of cell is selected from the range ofendocrine secretory cells, and is capable of expressing effectiveamounts of at least one secretion into the growth medium when inassociation with the second type of cell, so that the one or moresecretions may be isolated from time to time for use.

In a subsidiary aspect, the secreted product from a functionalartificial tissue the secretion includes an effective amount of insulin.

In a related aspect, the functional artificial tissue is preserved fortransport, storage and later use in a state of metabolic arrest.

In a further related aspect, the functional cells include cells capableof forming an extracellular matrix which may be shaped to suit a graftor transplant, including chondrocytes, osteocytes, fibrocytes fromtendons, and the skin.

Preferably the effective amount of serum from the same species of mammalis at least 5% and more preferably is about 10%.

Preferably the effective amount of nicotinamide is at least 5 mM andmore preferably is about 10 mM.

More preferably the invention provides an example tissue cultureenvironment comprising Sertoli cells as a food layer for islet cells inthe growth media known as RPMI 1640 media supplemented with 10% porcineserum, 10 mM nicotinamide, and 11.1 mM glucose (noting that thesenumbers are examples).

Preferably the Sertoli cells are from the same species of animal; morepreferably they are from the same animal.

Preferably a ratio of seeded Sertoli cells to seeded islet cells isabout 10,000 to 1 islet-equivalent (IEQ).

As alternatives to Sertoli cells as support/trophic cells, one may useother cells having similar characteristics; unmodified cells includecertain neuroglial cells, secretory cells including choroid plexusepithelial cells.

Otherwise, modified cells include cells modified by alteration of theirgenetic composition so that they secrete a range of compounds similar tothose secreted by Sertoli cells in a tissue culture environment.

In a second related aspect the invention provides functional cellcombinations as previously described in this section, which have beengrown together for at least 50 days in growth media of this type.

Alternatively the invention provides groups of islet cells which havebeen grown together for at least 30 days in growth media of this type.

In a third broad aspect the invention provides living material suitablefor use in an xenotransplant; the material including groups of isletcells grown in an environment including (a) nicotinamide, (b) homologous(same species) serum, and (c) Sertoli cells all in a mutually effectiveamount.

Preferably the living material is encapsulated and a preferredencapsulation is carried out according to the Calafiore method.

Optionally the living material is provided in a metabolically slowed, orpreserved state for transport or storage.

In a fourth broad aspect the invention provides a method of in vitropropagation and differentiation of functional islets in a medium aspreviously described in this section, using Sertoli cells as a matrixand a “nurse or feeder” layer.

Preferably the method includes the steps of

-   1. isolating Sertoli cells from a neonatal mammal-   2. isolating pancreatic islet cells or progenitors thereof from a    neonatal mammal,-   3. forming a cultured layer of Sertoli cells on a surface, using a    growth medium as herein described,-   4. adding islet cells-   5. and maintaining the cell combinations over a period, until the    islet cells have differentiated into functional cells, and formed    concentrated masses capable of secreting insulin at least partially    in response to a glucose stimulus.

In a fifth broad aspect, the invention provides a method for growingfunctional cells or cell associations in vivo and in a containerincluding a liquid medium; the medium being separated from the cells orcell associations from time to time so that the one or more desiredsecretions expressed by the cells may be harvested from the cells for apharmaceutical use.

Preferably a desired secretion is insulin.

Preferred Embodiment

The description of the invention to be provided herein is given purelyby way of example and is not to be taken in any way as limiting thescope or extent of the invention. The specific developments that havetaken place during development of this invention are in relation toxenotransplantation for cases of diabetes. However it must be realisedthat the Examples to follow can be applied to other types of cells, suchas hepatocytes, chondrocytes, and the like.

Illustrations

FIG. 1. Photomicrograph of a group of adjacent islets at various stagesof development. A cell monolayer forms a background to a lower focalconcentration of cells (normally after 5-7 days in culture), an upperfocal formation of three-dimensional structures (normally at 2-3 weeks),and in the centre an islet-like structure (normally at 34 weeks inculture).

FIG. 2: Islet cells (Liberase H isolation) cultured for 5 weeks withHSA.

FIG. 3: Islet cells (Liberase H isolation) cultured for 5 weeks with 10%porcine serum (higher magnification than FIG. 2).

FIG. 4: Islet cells (Collagenase P isolation) cultured for 5 weeks withHSA.

FIG. 5: As FIG. 4 but with 100% porcine serum.

FIG. 6: Sertoli cells cultured for 5 weeks with HSA.

FIG. 7: As FIG. 6 but with 10% porcine serum

FIG. 8: Islets with Sertoli cells 1:100 (1 islet equivalent to 100Sertoli cells) cultured for 5 weeks with HSA.

FIG. 9: As FIG. 8 but with 10% porcine serum.

FIG. 10: Islets with Sertoli cells 1:10,000 cultured for 5 weeks withHSA

FIG. 11: As FIG. 10 but with 10% porcine serum; resulting in largeislets.

FIG. 12: Islets/Sertoli cells (1:10,000) cultured for 5 weeks withporcine serum. Diameter of islets: 300 μm and 600 μm.

FIG. 13: As FIG. 12. Diameter of islet: 600 μm.

FIGS. 14-17: Comparison of sizes of islets from different cultures with10% porcine serum: FIG. 14: Islet (Collagenase P isolation) 250 μm, FIG.15: Islets/Sertoli 1:100=150 μm, FIG. 16: Islets/Sertoli 1:10,000=500μm. FIG. 17: Islets/Sertoli cells cultured for 46 days. Size of theislet is 400 μm.

FIG. 18: DTZ staining of Islets/Sertoli 1:10,000 with HSA (some positivestaining)

FIGS. 19 & 20: DTZ staining of Islets/Sertoli 1:10,000 with porcineserum; 85% of cells positive to stain.

FIG. 21: is a bar graph to show the insulin response of free isletsafter 46 days of culture and the effect of the presence of Sertoli cellsduring growth on the response.

FIG. 22: is a bar graph to show the insulin response of encapsulatedislets and the effect of the presence of porcine versus human serumduring culture on the response, using a SGS test.

FIG. 23: is a bar graph to show the insulin response of islet cellsgrown together with a variety of other cell types including thefibroblast line HEF312.

In general this invention relates to improvements in the preparation ofcells for transplantation.

One aspect of the improvements is the provision of better growth mediafor the cells during preparation for transplantation. Another aspect isthe provision of a second cell type to form a supporting co-culture forthe islet cells, and a preferred cell type exhibiting a combination ofsupport and trophic effects, as it shows (among other functions) withinthe testis, is the Sertoli cell.

EXAMPLE 1a

This Example relates to tissue culture methods for raising and maturingcells intended for xenotransplantation.

Porcine Islet Cell Isolation. Pancreatic islets from 7 day old pigletswere prepared following an adapted method from C. Ricordi (PancreaticIslet cell Isolation. Austin R.G. Landes Co. 99:112, 1992). Our methodincludes enzymatic digestion (collagenase, liberase), culture for threedays at 37 deg C. in RPMI 1640 containing 2% human serum albumin,Ciproxin and 10 mmol/L nicotinamide in an atmosphere of 5% CO₂ and 95%air. Viability was tested with DTZ staining and insulin release invitro.

Porcine Sertoli Cell Isolation Cell Cultures in Combination. Testiclesfrom the same piglets were collected in HBSS solution includingantibiotics. The isolation of Sertoli cells was done following theRajotte procedure with some modifications (Rajotte, Diabetes, Vol 46,February 1997 317-322). The testicles were cleaned, separated from thecapsule, and minced into 1 mm pieces with scissors. Digestion was doneusing DNA-ase and collagenase in a pre-heated waterbath at 37° C.Viability was tested using Sudan m and Trypan blue.

Cell Cultures in Combination. A set of flasks was maintained for 50days, using a variety of media as per the following list. The media waschanged weekly hence some components may change in concentration duringthe week. The base is RPMI 1640 media supplemented with either 2% HSA(human serum albumin or 100% porcine serum, 10 mM nicotinamide, and 11.1mM glucose, and the following combinations and variations were tested:

-   1. Control Islets with 2% HSA-   2. Islets with 10% porcine serum-   3. Control Sertoli cells with 2% of HSA.-   4. Sertoli with 10% porcine serum-   5. Control Islets/Sertoli 1:100 with 2% HSA.-   6. Islets/Sertoli 1:100 with 10% porcine serum-   7. Control Islets/Sertoli 1:10.000 with 20% HSA.-   8. Islets/Sertoli 1:10.000 with 10% porcine serum

Samples were taken at day 46 for static stimulation with glucose (SGS)(FIGS. 21-22) and viability tests with DTZ (FIGS. 18-20) and AO/PIstaining. Results are expressed through photomicrographs FIGS. 1-20.Summary FIG. 1 is a photomicrograph of a group of islets illustratingdifferent stages of development close to each other, A cell monolayerforms a background to a focal concentration of cells as in the lowerpart of the picture (normally seen after 5-7 days in culture). In theupper part of this photomicrograph there is an early stage of formationof three-dimensional structures (normally seen at 2-3 weeks), and in thecentre an islet-like structure (normally seen at 34 weeks in culture).

Compare FIG. 2 (islet cells (Liberase H isolation) cultured for 5 weekswith HSA) against FIG. 3 showing cells of the same origin cultured for 5weeks with 10% porcine serum (at a slightly higher magnification thanFIG. 2). An islet exists in the example where homologous (pig) serum isused. The human serum culture lacks evidence of growing cells.

Compare FIG. 4 (Islet cells (Collagenase P isolation) cultured for 5weeks with HSA) against FIG. 5—the same but with 10% porcine serum.Again, an islet is present with homologous serum while the human serumculture appears to be moribund.

Compare FIG. 6 of Sertoli cells cultured for 5 weeks with HSA in theabsence of islet cells against FIG. 7 (using 10% porcine serum); itappears that the homologous serum is beneficial to Sertolicells—preceding Figures also showed that homologous serum is beneficialto islet cells. The next set examines co-cultures.

Compare FIG. 8 Islets with Sertoli cells in the ratio of 1:100 (1 isletequivalent for every 100 Sertoli cells) cultured for 5 weeks using HSA,against FIG. 9 (using 10% porcine serum). There is some cell activity ina co-culture with HSA but islet formation is seen only within theporcine serum flask.

Using 1 islet equivalent (IEQ) for every 10,000 Sertoli cells appears tobe more optimal. In FIG. 10 that ratio with human serum resulted in noislet formation; whereas in FIG. 11, 10% porcine serum results in largeislets. Further examples using porcine serum at 10% are shown in FIGS.12 and 13. Diameter of islets: 300 μm, 600 μm, and 600 μm.

FIGS. 14 to 17 show various sizes of islets from differently treatedcultures all raised with 10% porcine serum: FIG. 14: Islet (CollagenaseP isolation) 250 μm, FIG. 15: Islets/Sertoli 1:100=150 μm, FIG. 16:Islets/Sertoli 1:10,000=500 μm. FIG. 17: Islets/Sertoli cells culturedfor 46 days and the size of this islet is 400 μm.

DTZ (dithizone) staining is shown in FIGS. 18, 19 and 20. FIG. 18 grownunder HSA (Islets/Sertoli 1:10,000) showed some positive staining. FIGS.19 and 20 are two examples of (the same co-culture ratio with 10%porcine serum. Here at least 85% of cells stained positively for thistest of insulin secretion.

One could conclude that there appears to be a synergistic co-operationbetween the use of homologous serum (in this case, 10% porcine serum)and co-culturing of islet cells with at least the Sertoli cells used inthis Example, because only when both components are present is thereformation of well-defined rounded islet structures as shown in many ofthe photomicrographs. This phenomenon could be described as“organogenesis”—if one can call an islet of Langerhans an organ. Itappears that diameters of about 600 microns is the upper limit of sizeat least for the protocols tested to date, and this may be related tooxygenation of the cells at the centre of each ball.

EXAMPLE 1b

Insulin release tests (SGS test; FIGS. 21 & 22). FIG. 21 is a bar graphto show the insulin response of free islets after 46 days of culture andthe effect of the presence of Sertoli cells during growth on theresponse. FIG. 22 is a bar graph to show the insulin response ofencapsulated islets and the effect of the presence of porcine versushuman serum during culture on the response.

All the cells used in FIG. 21 were cultured for 46 days in mediumsupplemented with 10% porcine serum. The maximal insulin release forfree islets at day 46 was 46.1 uU/100 IEQ/hr compared to 114 uU/100IEQ/hr when islets were co-incubated with Sertoli cells. Interestingly,the maximal insulin release was significantly higher when the mixture ofSertoli/islet cells was re-seeded one week before the SGS with a maximalinsulin release of 356.7 uU/100 IEQ/hr compared to 46.99 uU/100 IEQ/hrfrom re-seeded islets not as a co-culture. Interestingly, islet cells inco-culture with Sertoli cells were able to produce some insulin eventhough there were lot of apoptotic cells (see photo 18). These resultsconfirm that Sertoli cells preserve the beta-cell function.

The same islets were then encapsulated in alginate according to methodsdescribed by Calafiore et al (see Calafiore Basta & Boselli, TransplantProc 1997; 29: 2126-7, (“Effect of alginate/polyaminoacidic coherentmicrocapsules (CM) transplantation in adult pigs”) and Calafiore, Basta& Falorni, Diabetes Nutr Metab 1991; 4: 45-48 (“Vascular graft ofmicroencapsulated human pancreatic islets in non immunosuppresseddiabetic recipients; preliminary results”) and cultured with human orporcine serum. The insulin release results are shown in FIG. 22. “Staticstimulation Encapsulated Islets: Human vs Porcine serum”. The averagemaximal insulin release after 10 days was 54.57 uU/100 IEQ/hr for theencapsulated islets maintained with porcine serum compared to 128 uU/100IEQ/hr when cultured with human serum. However, after 36 days in cultureinsulin release decreased to 54 uU/100 IEQ/hr for encapsulated isletswith HAS and increased to 154 uU/100 IEQ/hr for encapsulated islets withporcine serum.

In order to demonstrate the effect of serum species on functional cellnumbers, see the photomicrograph FIG. 18: which is of a culture usingIslets/Sertoli cells in a ratio of 1:10.000 with HAS. Only some isletsstill show some positive DTZ staining. In comparison, DTZ staining inFIGS. 19 and 20 with the same ratio of cell types but with porcine serumshows that more then 85% of the cells are DTZ positive cells.

It should be noted that we did not seek an immunological barriercomprised of Sertoli cells.

EXAMPLE 1c DNA Flow Cytometry

A major concern for workers in this art is that the neonatally derivedcells may undergo a potentially malignant transformation. Thus, for anylong-term culture of cells-precursors the control of “normality” is veryimportant Flow cytometry has been used to check the ploidy of thecultured cells in this study. Table 1 represents the DNA contentfollowing different treatments. TABLE 1 DNA content in differentexperiment settings after 50 days in culture. Treatment\Resulting ploidyDiploid Tetraploid Aneuploid Islet cells with 2% HSA (no events) 0 0 0Islet cells (Liberase H isolation) 93.6% 4.91% 0.44% with 10% porcineserum Islet cells (Collagenase P isolation)   93%   5% 0.88% with 10%porcine serum Islet/Sertoli cells with 2% HSA 0.03%   0%   0%Islet/Sertoli cells with 10% porcine 88.2% 9.89%  0.9% serum PK15(porcine embryo kidney 66.6% 24.9% 3.72% cell line) as a same-speciescontrol

EXAMPLE 1d

This Example relates to another tissue co-culture method for raising andmaturing islets in combination with the recipient's own fibroblasts asobtained for example from a skin punch biopsy. Fibroblasts appear to beat least an equivalent to Sertoli cells in terms of a synergisticsupporting relationship, and the resulting cell groups are suitable forxenotransplantation. The recipient's fibroblasts (allocells) are used asa feeder layer and ideally would grow over the surface of a globule ofislet cells during tissue culture and form a surface layer capable of atleast partial immunoisolation so that the islet-fibroblast structurescan be put into the recipient for insulin production. This isolationshould prolong the effective life of the xenotransplant. This idea canbe extended to the usage of any other recipient cells such asendothelial cells, chondrocytes etc.

Experimental procedures. Porcine islets isolated according to example 1awere put in co-culture with human primary (non-transformed) fibroblastsas well as with other cell types to compare the extent of proliferationand both amount and responsiveness of insulin release. The followingcell types were used as a feeder layer:

-   1. Human embryonic kidney (HEK293) [ATCC CRL-1573]-   2. HeLa line-   3. Human fibroblasts (HEF312) [Auckland Hospital]-   4. Porcine islet cells-   5. Porcine Sertoli cells

Flasks with RPMI 1640 media supplemented with either 2% HSA or 10%porcine serum, 10 mM Nicotinamide, and 11.1 mM glucose were maintainedfor 40 days:

-   1. Control Islets/human fibroblasts with 2% H.S.A. (human serum    albumin)-   1. Islets/human fibroblasts with 10% porcine serum-   2. Control islets/Sertoli cells with 2% of H.S.A.-   3. Islets/Sertoli with 10% porcine serum-   4. Control Islets/HeLa with 2% H.S.A.-   5. Islets/HeLa with 10% porcine serum-   6. Control Islets/HEK293 with 2% H.S.A.-   7. Islets/HEK293 1:10.000 with 10% porcine serum

The media was changed twice per week. After 40 days in co-culture,assessment of results was carried out. Photomicrographs were taken.Samples were also taken at day 40 for static stimulation with glucose(SGS) and DTZ staining. As shown in FIG. ______ the best result from thestandardised SGS procedure was obtained from the islet/fibroblastco-culture, with about 1350 micro-units per 100 IEQ/hour, and the outputof insulin dropped subsequently. Although this Example has not includedthe initial step of obtaining a large number of fibroblasts from a skinbiopsy (we used a fibroblast line known as HEF312), the validity of theuse of fibroblasts has been demonstrated.

Variations

This Example may also relate to tissue culture methods for raising andbringing to functional maturity various cells and functional cellgroups, other than islet cells, which are intended forxenotransplantation.

Note that the concentrations of porcine serum have been simply set at10% without any attempt so far to ascertain an optimum amount.Nevertheless this Example covers such variations, just as it coversother concentrations of nicotinamide apart from the specified 10 mM.Further, the amount of glucose in the culture media used has not yetbeen the subject of experiment.

Apart from Sertoli cells, known to have an immunologically privilegedposition, it may be possible to use as trophic and supportive cellsother types of cells including genetically modified cells, andunmodified neural support (neuroglial) cells, fibroblasts, adult stemcells, and chondrocytes.

Indeed, Example 1d supports the use of fibroblasts. There may be furtherbenefit in using mixtures of Sertoli (or other types of) cells andfibroblasts together with porcine islet cells.

It may become ethically acceptable to maintain, for an individual, abank of “spare cells” preserved in case of later need. This mightinclude individuals at the onset of clinical diabetes, or individualshaving a known susceptibility to a disease. It may also includehomograft techniques wherein human cells (that is, Sertoli and isletcells or precursors thereof) are raised together, if needed, in thepresence of human serum and the resulting islet cell groups may beencapsulated and returned to the individual. Incidentally the Sertolicells may be from a different individual because it is unlikely thatSertoli cells will be included within the masses of islet cells that areencapsulated and used in transplants.

Commercial Benefits or Advantages

Growing of animal cells in vitro for xenotransplantation (as compared toharvesting islets from mature animal pancreases) facilitates theprocedure of encapsulated islet preparation, and would allow bettercontrol of infectious pathogens such as potentially zoonotic viruses,even retroviruses.

The invention also provides for the manufacture of cell secretions basedon in vitro preparations, as an alternative to recombinant or syntheticmanufacture. The advantage being that any glycosylation or the like ofthe peptide or proteins produced is provided by the mammalian cellsthemselves, and that any contaminating micro-organisms may be excludedfrom the products.

This invention provides a supply of islet cell groups suitable forencapsulation by known means and then for use in xenotransplants; theadvantage of this invention being that the cell groups are larger, moreactive and available in greater quantities than was formerly possible.

Finally, it will be understood that the scope of this invention asdescribed and/or illustrated within this provisional specification isnot limited to the preferred embodiments described herein forillustrative purposes. Those of skill will appreciate that variousmodifications, additions, and substitutions are possible withoutdeparting from the scope and spirit of the invention as set forth in thefollowing claims.

1. A method for carrying out long-term in vitro assembly, propagationand maturation of a functional artificial tissue comprised of more thanone kind of cell taken from at least one species of mammal,characterised in that a selected first type of cell capable of beingstimulated to secrete a product is co-cultured over an extended periodtogether with at least one selected second type of cell capable ofproviding a support/trophic function for the first type, in a growthmedium supplemented with an effective amount of homologous serum fromthe species of mammal that provided the first type of cell, so thatorganised functional, synergistic groups of mixed cell types aredeveloped over the extended period.
 2. A method as claimed in to claim 1for making a functional artificial tissue characterised in that theartificial tissue includes a first type of cell and a second type ofcell jointly capable of possessing a functional property on beingco-cultured in a growth media supplemented with an effective amount ofhomologous serum, so that the artificial tissue may be grafted into arecipient suffering from a deficiency of the property, to alleviate thedeficiency.
 3. A method as claimed in claim 1, characterised in that thefirst type of cell is selected from the group of endocrine secretorycells and the product is an endocrine hormone.
 4. A method as claimed inclaim 3, characterised in that the second type of cell is selected froma group including Sertoli cells and fibroblasts.
 5. A method as claimedin claim 1, characterised in that the effective amount of homologousserum is in a range of from about 5% to about 15% by volume in thegrowth medium.
 6. A method as claimed in claim 1, characterised in thatthe growth medium also includes nicotinamide within a range of fromabout 5 mM to about 15 mM.
 7. A method as claimed in claim 1,characterised in that at least the first type of cell is collected froma neonatal mammal.
 8. A method as claimed in claim 4 for making afunctional artificial tissue characterised in that the first type ofcell is endocrine cells from the pancreatic islets of Langerhans andthat a ratio of the first type of cell to the second type of cell isoptimised.
 9. A method as claimed in claim 8 for making an artificialtissue characterised in that the artificial tissue is grown as aco-culture in a growth media supplemented with an effective amount ofhomologous serum, glucose, and nicotinamide, so that the resultingartificial tissue exhibits a functional secretory response to a localconcentration of glucose, so that the artificial tissue may be graftedinto a recipient suffering from diabetes in order to alleviate thedisease.
 10. A method as claimed in claim 9 further characterised inthat the second type of cell is taken from an individual in need of afunctional graft and after in vitro propagation and maturation togetherwith the first type of cell for a period is returned to the diabeticrecipient in the form of a functional artificial tissue.
 11. A method asclaimed in claim 10 further characterised in that the functional cellcombinations have been grown as a co-culture for from about 30 to about50 days in growth media of this type.
 12. A functional artificial tissuecomprised of more than one kind of cell taken from at least one speciesof mammal, characterised in that the functional artificial tissue iscomprised of at least partially organised synergistic accumulations ofcells comprised of a first type of cell capable of being stimulated tosecrete a product, and at least one second type of cell capable ofproviding a support/trophic function for the first type of cell, thefunctional artificial tissue being formed by co-culture in vitro over anextended period of time.
 13. A functional artificial tissue as claimedin claim 12, characterised in that the first type of cell is selectedfrom the range of endocrine secretory cells, and is capable ofexpressing effective amounts of at least one secretion into the growthmedium when in association with the second type of cell, so that the oneor more secretions may be isolated from time to time for use.
 14. Asecreted product from a functional artificial tissue as claimed in claim13, characterised in that the secretion is insulin.
 15. A functionalartificial tissue as claimed in claim 12, characterised in that thefunctional artificial tissue is preserved in a state of metabolic arrestsuitable for transport storage and later use.